Adult male Sprague-Dawley rats (220g-250g;) were purchased from the Army Medical university and kept on a feedback-controlled heating pad to maintain body temperature at 37.0℃. Animal using procedures was in compliance with the guide for the care and use of laboratory animals and approved by the animal care and use committee at the Army Medical university (SCXK-PLA-20120011).
ICH-IVH Model of hydrocephalus and drug treatment
The surgical procedures for rats subjected to hemorrhage were well described in our previous study[9, 15]. In brief, animals were anesthetized with pentobarbital (40 mg/kg intraperitoneal). The right femoral artery was catheterized as a source of blood sample. The rats were positioned in a stereotaxic frame, a cranial burr hole (1 mm) was drilled (coordinates:0.2 mm posterior and 2.2 mm lateral to the bregma). A 29-gauge needle was inserted at a rate of 1mm/min at the depth of 5.0 mm from the dura. Using a microinjection pump, 200μl nonheparinized arterial blood was infused into the right caudate nucleus at a rate of 14μl/min through the hole. Normal saline was infused as vehicle groups. NLRP3 inhibitor MCC950 (MCE, USA) dissolved in normal saline was administered by intraperitoneal injection 1 hour after ICH-IVH at a dose of 10mg/kg. Bumetanide (MCE, USA) dissolved in normal saline was administered by intraperitoneal injection each day after ICH-IVH at a dose of 10mg/kg.
Brain water content
Three days after blood infusion, rats were euthanized without perfusion. Brains were removed immediately and divided into three parts: left-hemisphere, right-hemisphere and cerebellum. Firstly, the wet weights were measured using fresh tissue. Then, the brain sections were dried at 100℃ for 24 hours before obtaining the dry weights. The percentage brain water content was calculated as follows formula: (wet weight–dry weight)/wet weight100.
Neurological dysfunction of rats was evaluated using a modified Neurological Severity Score (mNSS) method as described previously. Briefly, the assessment was performed at 3 and 7 days after ICH-IVH. First, the mNSS are a composite test of motor, sensory, and balance functions. Neurological function was graded on a scale of 0-18 (a score of 13-18 indicates severe injury, 7-12 indicates moderate injury, and 1-6 indicates mild injury).
Qualification of CSF secretion rate
Rates of CSF production were measured using the method as previously described. Briefly, anesthetized rats were mounted on a stereotactic apparatus, and a cranial burr hole (1.2 mm) was drilled over the left lateral ventricle (coordinates, relative to the bregma: x, -0.6cm; y, -1.6cm). Next, the rat’s head was rotated on the ear-bars by 90° so that it was oriented nose down, and the suboccipital muscles were dissected to the cisterna magna to expose the atlantooccipital ligament. The ligament was punctured, and a 29-gauge needle was advanced 5 mm through the foramen of Magendie to the fourth ventricle. Sterile molecular-grade oil (100μl; Sigma-Aldrich) was infused through the tube to occlude the aqueduct of sylvius, thereby creating a closed system of CSF circulation in lateral ventricle. With the rat in the same position, a glass capillary tube (OD,1.1mm; ID,1.0mm; length,20cm) was advanced through the burr hole into the lateral ventricle (depth 4.5mm ventral). The volume of CSF that collected at a given time (20 min) was calculated as: V (mm3) =π·r2·d, where r is the radius of the glass capillary tube and d is the distance that CSF traveled within the capillary. The rate of CSF formation (μl/min) could be calculated from the slope of volume-time relationship.
Transmission electron microscope
Electron microscopy was performed as previously described. Rats were anesthetized and subjected to intracardiac perfusion with 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1mol/L Sorensen’s buffer (pH 7.4). The choroid plexus were removed from brain and a 1-mm-thick coronal tissue slice was cut with a blade 4 mm overnight at 4°C. Samples were then post-fixed with 1.0% OsO4 and dehydrated in graded ethyl alcohol. After completion of dehydration, samples were infiltrated with propylene oxide, embedded in Epon resin, and sectioned. Ultra-thin sections were then stained with uranyl acetate and Reynold’s lead citrate. Sections were evaluated using a Philips CM 100 transmission electron microscope (Hillsboro, OR, USA) and digitally acquired using a Hamamatsu (Hamamatsu City, Shizuoka, Japan) ORCA-HR camera.
Blood-CSF Barrier permeability measurement
CSF was obtained at 3 days after blood infusion by puncture of the cisterna magna and stored at -80℃ prior to analysis, and dextran-10,000 MW (Invitrogen, USA) was given by vein 2 hours before collecting CSF. CSF was measured absorbance at 540nm using Spectrophotometer.
Western blot analysis
Western blot analysis was performed as previously described. The brains were perfused with saline before decapitation at day 3 after injection. The choroid plexus tissue was sampled. The following primary antibodies were used: rabbit anti-NLRP3 (1:1000 dilution, Abcam, UK), mouse anti-Caspase-1 (1:1000 dilution, Novus, USA), rabbit anti-IL-1beta (1:1000 dilution, Gene-Tex, USA), rabbit anti-p-NKCC1 (1:1000 dilution, Sigma-Aldrich, USA) , rabbit anti-NKCC1 (1:1000 dilution, CST, USA), rabbit anti-ZO-1 (1:5000 dilution, Abcam, UK) ,and rabbit anti-β-actin(1:1000 dilution, CST, USA). The relative densities of the immune-reactive bands were normalized to β-actin and analyzed using ImageJ (National Institutes of Health, Bethesda, Maryland, USA).
Cytokines Assay Kits
Rats were anaesthetized with pentobarbital (40 mg/kg intraperitoneal). CSF was collected through the cisterna magna, then rats were decapitated to get the choroid plexus. The CSF was centrifuged at 10,000 rpm to separate the supernatant. The choroid plexus were homogenized in ice-cold PBS with protease inhibitor cocktails (Sigma-Aldrich, USA). Total protein concentration was quantified by the BCA protein assay (Boster, China). The CSF and choroid plexus cytokines levels were measured using inflammation cytokines assay kits (Raybiotech, USA) according to the manufacturer’s instructions.
Immunofluorescence staining of brain tissue was performed on fixed frozen sections as previously described. Rats were anesthetized with pentobarbital (40 mg/kg intraperitoneal) and perfused with 4% paraformaldehyde for 4 to 6 hours. The brain was isolated and then dissected the choroid plexus under magnification using sharp forceps. The choroid plexus were immersed in 30% sucrose for 3 to 4 days at 4℃. The tissues were embedded in an optimal cutting temperature compound (SAKURA, USA), and 12-mm-thick slices were cut using a cryostat. The slices were incubated with the following primary antibodies at 4℃ overnight: rabbit antibody to NLRP3(1:200, Abcam, UK), rabbit antibody to p-NKCC1(1:200 dilution, Sigma-Aldrich, USA). Then the slices were probed with appropriate secondary antibodies for 2 h at 37 °C. Finally, the slices were counterstained with DAPI (Boster, China) and examined using a confocal fluorescence micro- scope (LSM780, Zeiss).
The cell counts were performed at day 3 after ICH-IVH, respectively. Cell counts analysis was performed as previously described. For quantification of the positive cells in the choroid plexus, consecutive slices were made, and two sections per animal (n=6 per group) with 40-μm space in between were used for cell counts. Three high-power images were used for cell counting. NLRP3 and p-NKCC1 positive cells were counted in the choroid plexus. Cell counts were performed by two researchers in a blinded manner. All measurements were repeated three times, and the mean value were used.
MRI and ventricular volume analysis
Rats were anesthetized with 2% isoflurane/air mixture throughout MRI examination. The MRI scans were performed in a 7.0-T Varian MR scanner (Bruker, USA) with a T2*gradient-echo sequence and a T2 fasts spin-echo sequence using a view field of 35mm*35mm and 17 coronal slices (1.0mm thickness). Volumes were calculated as previously described. Bilateral ventricles and hippocampus were outlined, and the areas of all slices and multiplying by the section thickness. All image analyses were performed using Image J (National Institutes of Health, Bethesda, Maryland, USA) by two observers in blinded manner.
The values in this study are presented as mean ± SD. SPSS 19.0 software was used to analysis all data. Data were analyzed by Student t test for single comparisons or ANOVA with post hoc Bonferroni-Dunn correction for multiple comparisons. A P value of <0.05 was considered statistically significant.